The initial interactions of viral envelope proteins with host cells are critical for the establishment of infection by most animal viruses. Invertebrates serve as intermediate hosts for numerous viral pathogens of humans, but relatively little is known about the interactions between viruses and invertebrate cells. Baculoviruses are large DNA viruses of insects and as such, serve as an important model system for studies of the interactions of viruses with invertebrate cells. The baculovirus envelope glycoprotein, gp64, is a neutralizing antigen and shows striking amino acid sequence identity with the envelope proteins of two tick vectored orthomyxo-like viruses (Thogoto and Dhori arboviruses) of vertebrates. This indicates that these arboviruses and baculoviruses share a common mechanism for infecting invertebrate cells. However, these baculovirus and arbovirus envelope proteins show no apparent sequence or structural similarity to the HA proteins of orthomyxoviruses, such as influenza. Recent studies (this laboratory) show that the baculovirus gp64 protein is a pH dependent membrane fusion protein, indicating that gp64 mediates fusion of the virion envelope with the endosome membrane during virion entry by endocytosis. In the proposed studies, the function of the gp64 protein will be examined in detail by multiple and complementary methods. Protein functions and functional domains will be identified and characterized with a specific focus on membrane fusion and cell receptor recognition. The following approaches will be used: 1) Directed mutagenesis of the protein and functional assays will identify functional domains of the protein. 2) Anti-gp64 monoclonal antibodies (MAbs) will be generated and screened for neutralization of membrane fusion or virion infectivity. Epitopes recognized by neutralizing MAbs will be mapped. 3) Recombinant baculoviruses containing discrete mutations within structural and functional domains of the gp64 protein will be generated and used to examine the effects on viral infectivity, the infection cycle, and virion maturation. 4) Cellular receptors that interact with the gp64 protein (or other proteins on the budded virus) will be identified, cloned, and sequenced. Approaches similar to those described above (1 and 2) will be utilized to identify domains required for binding to viral envelope proteins. In total, these studies will provide valuable new insight into the highly conserved molecular mechanisms used by invertebrate viruses to recognize and enter host cells.